Wnt signaling is one of the key pathways controlling growth and patterning of the vertebrate central nervous system. Mutations in components of the Wnt signaling pathway often lead to birth defects and tumors affecting the nervous system. Thus, understanding the mechanisms of Wnt regulation in the context of nervous system development is vital for our understanding and treatment of disease. The focus of this study is to elucidate the role of Oto, a novel regulator of Wnt proteins, in spinal cord development and the Wnt signaling pathway. The otocephaly mutant has a variety of phenotypes that appear to be due to either excess or early onset of Wnt signaling, including holoprosenchephaly, agnathia and spinal cord overgrowth. The Oto locus encodes an enzyme required for proper glycosylphosphatidylinositol (GPI)-modification of Wnts. This provides potential new insights into the mechanisms of Wnt regulation. Wnts, which are secreted and considered to be long-range signaling molecules, may be GPI-anchored to lipid membranes in some physiological conditions. GPI-linked proteins are targeted to lipid rafts and segregate from the general population of nonGPI-linked proteins in the secretory pathway. The complex biology conferred by GPI modification provides multiple points of regulation for the activity of GPI-linked Wnts. I hypothesize that Oto functions to regulate cellular sorting, secretion and/or tissue mobility of Wnts, thus regulating the availability and/or signaling efficiency of Wnts. To address this hypothesis, I will examine Oto function at multiple levels. I will examine well-described features of spinal cord development known to be under Wnt control, including progenitor proliferation and dorsal-ventral patterning, and correlate potential findings to changes in Wnt signal transduction. I will also examine the function of Oto in the maturation of Wnt proteins through the secretory pathway in cultured cell systems. Furthermore, I will directly analyze the function of Oto in the establishment of chick Wnt1 protein distribution in the chick spinal cord. For the spinal cord experiments, both mouse genetics and in ovo chick embryo electroporation will be utilized. The proposed studies should expand our understanding of spinal cord development and Wnt signaling regulation. A number of human diseases and malformations are associated with developmental defects in the Wnt pathway, including holoprosencephaly, brain tumors and neural tube defects. Many of these syndromes have a genetic basis in mutations affecting the Wnt pathway, but teratogenic effects of drugs augmenting the Wnt pathway are also an important cause of neural tube defects in humans. This proposal will allow new understanding of the molecular and developmental events causing these important syndromes.